**6.3. CYP1A1 and CYP2B in different species after treatment with flame retardants**

58 New Approaches to the Study of Marine Mammals

induce this cytochrome.

treatments.

The results confirm the capability of this methodology to detect CYP1A1 (Table 2A) and CYP2B (Table 2B) induction with OC mixture in many species of this study; particularly we had induction of CYP1A1 and CYP2B, with respect to chemical blank (DMSO), at all doses in fin whale (RT2) (Figure 3A; D) and long-beaked common dolphin (MDC12); an induction of CYP1A1 was detected at all doses in Risso's dolphin (GGL1) and of CYP2B at all doses in sperm whale (PM6), killer whale (MOO12) and striped dolphin (RT1) (Figure 3F). No induction of CYP1A1 was detected in sperm whale (PM6) and bottlenose dolphin (TurNic), while CYP2B showed OC induction at least at one treatment dose in all species. Different induction responses were given by the different specimens: there was a dose/response induction for CYP1A1 only for long-beaked common dolphin (MDC12) and for CYP2B only for fin whale (RT2) (Figure 3D), while a bell-shaped response was present for CYP1A1 in Risso's dolphin (GGL1) and common dolphin (DDL1), and for CYP2B in sperm whale (PM6), killer whale (MOO12) (Figure 3E), striped dolphin (RT1) (Figure 3F) and common dolphin (DDL1). Discontinuous induction response was showed for CYP1A1 and CYP2B by the other specimens such as CYP1A1 in killer whale (MOO12) (Figure 3B) and striped dolphin (RT1) (Figure 3C). It is interesting to point out that all species, following treatment with the OC mixture, showed a greater response of CYP2B, compared to CYP1A1, confirming that these xenobiotics mostly with globular structure have a major ability to

**Figure 3.** A-F: Immunofluorescence (AUF/nucleus) of CYP1A1 (A-C) and CYP2B (D-F) in fibroblast cells of fin whale (RT2) (A, D), killer whale (MOO12) (B, E) and striped dolphin (RT1) (C, F) treated with OC mixture. DAPI and Alexa Fluor 594 (Intensity 200ms) images of DMSO and the five OC mixture

Results of the mean levels of immunofluorescence of CYP1A1 (A) and CYP2B (B), revealed in cultured fibroblasts of different species treated with flame retardants and expressed as index numbers, are reported in Table 3A-B. Marked differences in CYP1A1 (Table 3A) and CYP2B (Table 3B) induction by flame retardants were detected in different species, with higher sensitivity of responses in striped dolphin (RT1) for CYP1A1 (Figure 4A) and killer whale (MOO12) for CYP2B. To be highlighted that we have an inductive response of both cytochromes in the same animals, precisely in sperm whale (PM6), killer whale (MOO12), striped dolphin (RT1) (Figure 4A; C), long-beaked common dolphin (MDC12) (Figure 4B; D) and common dolphin (DDL1). Bottlenose dolphin (TurNic) showed only the CYP1A1 induction.


**Table 3.** (A-B): Mean values of immunofluorescence of CYP1A1 (A) and CYP2B (B) revealed in cultured fibroblasts of different species treated with flame retardants. The immunofluorescence is expressed as index numbers respect to solvent control. Different colour of box is related to different increase of these proteins. N.C. = no cells.

A dose dependent induction of CYP1A1 was detected in striped dolphin (RT1) and common dolphin (DDL1) and of CYP2B only in long-beaked common dolphin (MDC12). A bellshaped response was present for CYP1A1 and CYP2B in sperm whale (PM6), and for CYP2B

in killer whale (MOO12) and striped dolphin (RT1), while discontinuous induction responses were showed for CYP1A1 and CYP2B by the other species. Also these contaminants are mainly with globular structure as OC mixture, but no differences are present in the induction of the two cytochromes.

"Test Tube Cetaceans": From the Evaluation of Susceptibility to the Study of

*0.1% Dose C Dose B Dose A* 

*0.1% 0.01 µg/ml 0. 1 µg/ml 1 µg/ml* 

Genotoxic Effects of Different Environmental Contaminants Using Cetacean Fibroblast Cell Cultures 61

assessed with trypan blue, a quality control test to check the cell preparation. In the event of cell damage, trypan blue penetrates the cell membrane, and dead or damaged cells appear blue [36]. CYP2B also, showed induction in the three species treated with PAHs (Table 4B). The striped dolphin (RT1) showed the same trend for the two cytochromes (Table 4A-B).

> **MBE3 (Bryde's whale)** 100 31.6 106.2 55.8 **PM6 (sperm whale)** 100 111.5 69.6 68.2 **GGL1 (Risso's dolphin)** 100 107.7 90.2 D.C. **RT1 (striped dolphin)** 100 279.8 77.3 D.C.

> **MBE3 (Bryde's whale)** N.C. N.C. N.C. N.C. **PM6 (sperm whale)** 100 125.0 64.2 101.6 **GGL1 (Risso's dolphin)** N.C. N.C. N.C. N.C. **RT1 (striped dolphin)** 100 108.6 25.3 D.C.

**MDC12 (long-beaked common dolphin)** 100 155.6 279.7 123.2

**MDC12 (long-beaked common dolphin)** 100 218.6 65.6 64.3 **Table 4.** (A-B): Mean values of immunofluorescence of CYP1A1 (A) and CYP2B (B) revealed in cultured fibroblasts of different species treated with PAHs (Dose C = 0.5μM BaP + 10μM BnF, Dose B = 2.5μM BaP + 50μM BnF and Dose A = 12.5μM BaP + 250μM BnF). The immunofluorescence is expressed as index numbers respect to solvent control. Different colour of box is related to different increase of these

In Table 5A-B we reported the results of the mean levels of immunofluorescence of CYP1A1 (A) and CYP2B (B), revealed in cultured fibroblasts of killer whale (MOO12) treated with

**MOO12 (killer whale)** 100 800.7 886.3 747.3 D.C.

**MOO12 (killer whale)** 100 48.3 40.4 49.2 D.C. **Table 5.** (A-B): Mean values of immunofluorescence of CYP1A1 (A) and CYP2B (B) revealed in cultured fibroblasts of different species treated with BPA. The immunofluorescence is expressed as index numbers respect to solvent control. Different colour of box is related to different increase of these

Only one species, the killer whale, was treated with this potent estrogenic compound since the study to evaluate the cetacean susceptibility to BPA has just started in our laboratories. However, this preliminary experiment confirms that BPA is a potent CYP2B inhibitor [35]

*A CYP1A1 Ethanol 0.1% 0.1 µg/ml 1 µg/ml 10 µg/ml 100 µg/ml* 

*B CYP2B Ethanol 0.1% 0.1 µg/ml 1 µg/ml 10 µg/ml 100 µg/ml* 

**6.5. CYP1A1 and CYP2B in different species after treatment with BPA** 

*A CYP1A1 Acetone* 

*B CYP2B DMSO* 

proteins. N.C. = no cells. D.C. = death cells.

BPA, expressed as index numbers.

proteins. D.C. = death cells.

**Figure 4.** A-D: Immunofluorescence (AUF/nucleus) of CYP1A1 (A-B) and CYP2B (C-D) in fibroblast cells of striped dolphin (RT1) (A, C) and long-beaked common dolphin (MDC12) (B, D) treated with flame retardants. DAPI and Alexa Fluor 594 (Intensity 200ms) images of Nonane and the three flame retardant treatments.
